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This part of the project aims to describe changes in markers of vascular competence (Endothelial Microparticles Platelet (EMP), Circulating endothelial cells (CEC) and Circulating endothelial progenitors (EPC)) at different ages of lifespan both in physiological state and in response to hypoxia-induced vascular stress.
Full description
Blood will be harvested in citrated or EDTA anticoagulant and processed within one hour for Endothelial Microparticles Platelet (EMP), Circulating endothelial cells (CEC) and Circulating endothelial progenitors (EPC) determination.
Methods for endothelial marker determination:
Endothelial Microparticles Platelet free plasma (PFP) will be prepared by a double step centrifugation of citrated blood at 1,500g for 15 min and 13,000 g for 2 min at room temperature. Then EMP will be enumerated by flow cytometry after labelling of PFP using monoclonal antibodies directed against endothelial antigens, such as CD144 (19). To investigate the proportion of EMP within the whole circulating MP, MP for platelet, leukocyte and erythrocyte origins will be determined using respectively, CD41, CD45, Glycophorin-directed antibodies, and the total number of phosphatidylserine expressing MP will be determined using AnnexinV binding.
CEC will be enumerated using the consensual methodology based on an immunomagnetic separation assay (20). CEC will be isolated from EDTA whole blood using magnetic beads coated with antibodies directed against the CD146 antigen. Isolated cells will be identify using additional criteria such as morphology, size higher than 15µm, cell rosettes bearing more than 5 beads, and expression of endothelial markers (lectin binding).
For EPC two complementary approaches will be used for determination:
We designed a protocol which specific aims are 1/ to test the effect of local hypoxia on the markers of endothelial damage/repair equilibrium 2/ to define whether in elderly patients, local ischemia can be used to mobilize EPC and to increase their proliferation potential compared to EPC isolated in resting conditions. Two groups of healthy volunteers aged 20-30 and 60-70 years from our cohorts will be submitted to forearm transient ischemia as described by Friedrich et al (23). Peripheral blood samples will be obtained before and after 10 min of venous occlusion performed with a cuff pressure midway between systolic and diastolic pressure (24). EPC, CEC and EMP will be enumerated according to the methods described supra and EPC will be tested for proliferating capacity at each time point. The arterial phenotype of conducting arteries will be evaluated, by the acute flow-mediated vasodilatation (FMD) and endothelium-independent vasodilatation (EIV) of the brachial artery (BA) in young and older healthy volunteers using a high-resolution echotracking system. The efficiency of venous occlusion will be evaluated by the release of t-PA by endothelial cells.
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